Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-03 to 2015-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

EC Number:

919-489-5

Molecular formula:

C13H23N

IUPAC Name:

Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200- SIT Kit
- Tissue batch number: Lot number: 21680
- Arrival date: 2015-07-14
- Date of initiation of testing: 2015-07-14 (pre-incubation)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 minutes at 37 ± 1.5 °C, 25 min at room temperature
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: at least 15 times, volume not provided
- Observable damage in the tissue due to washing: no data provided
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm
- Filter: 570 ± 1 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean tissue viability is ≤ 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is > 50 %

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: due to purity adjustment 30.4 μL (47 μL/cm2)
- Concentration: unchanged

NEGATIVE CONTROL
- Amount applied: 30 μL DPBS

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5 % SLS solution in deionised water

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

post-treatment: 42 h
MTT: 3 h treatment and 70 h extraction

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

90.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100.0 %

Positive controls validity:

valid

Remarks:

6.8 %

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. The test substance is no MTT reducer.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. The test substance does not interfer with the color.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.
- Acceptance criteria met for positive control: Yes. Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 6.8% thus ensuring the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: Yes. The relative standard deviations between the % variabilities of the test item, the positive and negative controls in the main test were below 16 %, thus ensuring the validity of the study.

Any other information on results incl. tables

Dose Group	Exposure Interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Rel. Absorbance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Mean rel. Absorbance [% of Negative Control]***
Negative control	1 h	1.751	1.579	1.844	1.724	101.5 91.5 106.9	7.8	100.0
Positive control	1 h	0.129	0.115	0.109	0.118	7.5 6.7 6.3	8.6	6.8
Test item	1 h	1.862	1.860	1.923	1.881	85.5 79.6 106.8	15.8	90.6

* Mean of three replicate wells after blank correction

** relative absorbance [rounded values]: (100x(absorbance tissue)/ mean absorbance negative control

*** relative absorbance [rounded values]: (100x(mean absorbance test item/positive control))/ mean absorbance negative control

 

The mean relative absorbance value of the test item, corresponding to the cell viability,

decreased to 90.6% (threshold for irritancy:≤50%), consequently the test item was not irritant to skin.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was determined not to be irritating in the in vitro human skin model test with EpiDerm.

Executive summary:

An in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test according to OECD 439. The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary. Each three tissues of the human skin model EpiDerm were treated with the test item, the negative or the positive control for 60 minutes. 30.4 μL of the test item were applied to each tissue, and spread to match the surface of the tissue. 30 μL of either the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue. After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system. After treatment with the test item the mean relative absorbance value decreased to 90.6 % compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50 %. Therefore, the test item is not considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the substance is not irritating to skin.